Atherosclerosis, which is regulated by inflammation, endothelial cell (EC) barrier dysfunction, and oxidative stress, is the major cause of aortic aneurysm (AA), and mortality in Veteran population. Copper (Cu), an essential nutrient, is involved in normal function while excess Cu contributes to inflammatory diseases including atherosclerosis with unknown mechanism. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu transporting ATPase (ATP7A) which obtains Cu from cytosolic Cu chaperone Atox1 and then delivers Cu to secretory Cu enzymes including ecSOD, or exports Cu to extracellular space. Our lab reported that vascular ATP7A-ecSOD axis protects against endothelial dysfunction in hypertension and type1 diabetes, and that Atox1 functions as a Cu-dependent transcription factor, when it localizes at nucleus. However, a role of ATP7A and its relationship with nuclear Atox1 in atherosclerosis is entirely unknown. Preliminary data suggest that ATP7A mutant (ATP7Amut) mice which have reduced Cu export function crossed with ApoE-/- mice accelerate atherosclerotic lesion and AA with excess inflammatory cells and vascular permeability vs. ApoE-/- mice. By contrast, Atox1-/-/ApoE-/- mice exhibit significant reduction of atherosclerotic lesion, suggesting that ATP7A is atheroprotective while Atox1 is proatherogenic. Of note, both ATP7Amut and Atox1-/- mice show the similar extent of reduced Cu enzyme lysyl oxidase activity mediated through Cu chaperone Atox1, which does not explain enhancing atherosclerosis or AA in ATP7Amut/ApoE-/- mice. Based on additional new preliminary data with ATP7A-depleted ECs, we will test the novel hypothesis that Cu exporter ATP7A downregulation by inflammation increases intracellular Cu that stimulates nuclear Atox1- mediated EC barrier dysfunction and ROS-dependent inflammatory responses. This in turn promotes excess inflammatory cell recruitment, which accelerates atherosclerosis and AA. Aim 1 will define the protective role of ATP7A against inflammation-induced endothelial barrier dysfunction and ROS-dependent inflammatory adhesion molecule expression, which contribute to leukocyte transendothelial migration (TEM) in ECs in a Cu- and Atox1-dependent manner. Aim 2 will determine how inflammation-induced accumulated intracellular Cu promotes endothelial barrier dysfunction and ROS-dependent inflammatory responses by focusing on Cu-dependent transcription factor function of Atox1 to upregulate miR125b that represses VE- cadherin as well as p47phox of NADPH oxidase that increases ROS-NFkB. Aim 3 will define the protective role of ATP7A against atherosclerosis and AA development via regulating vascular permeability and inflammation in a Cu- and Atox1-dependent manner in vivo. We will use ATP7Amut, ATP7A transgenic, Atox1-/- mice; or inducible EC-specific ATP7A conditional deficient mice crossed with ApoE-/- mice with high fat diet treated with Cu chelators or LNA-anti-miR125b. Moreover, Cu imaging analysis (64Cu labeling, inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence microscopy (XFM)), live cell imaging, in vivo intravital microscopy, non-invasive Bioluminescence imaging using NFkB transgenic reporter mice will be used. Our study will provide novel insight into Cu transporter ATP7A or nuclear Atox1 as potential therapeutic targets for treatment of vascular inflammatory diseases such as atherosclerosis.